CN112795571B - Herbicide-resistant corn transformant and preparation method thereof - Google Patents

Abstract

Herbicide-resistant corn transformants are provided, along with related methods of development, detection, and applications.

Description

Herbicide-resistant corn transformant and preparation method thereof

Technical Field

The application relates to the technical field of plant biology, in particular to a method for creating a corn transformant capable of resisting herbicide glufosinate-ammonium and glyphosate, a detection method and application.

Background

Corn is an important feed and industrial raw crop, which is a crop with the largest planting area in China and has a long-term self-sufficiency, but the import amount is increased year by year since 2010.

The area of crops seriously damaged by weeds throughout the year in China is up to 12 hundred million acres, wherein the corn area is 1.9 hundred million acres. The weeds in the field compete with crops for water, fertilizer, light energy and growth space, are intermediate hosts of germs and pests which harm the crops, and are one of important biological limiting factors for increasing the yield of the crops. At present, the widely used selective herbicide has large application amount and long residual period, and is easy to influence the normal growth of the next crop. The biocidal herbicides such as glufosinate-ammonium and the like have the characteristics of high efficiency, low toxicity, easy degradation, no residue and the like, but have no selectivity in weeding and cannot be directly used in the growth period of crops. The plant transgenic breeding technology has the advantages of strong purposiveness, short period, high efficiency, capability of realizing the transfer of excellent genes among different species and the like. Since the first commercialization of transgenic crops in 1996, this technology has brought about tremendous changes to global agriculture.

In order to delay the resistance of insect-resistant corn to target insects, it is common practice to mix insect-resistant corn and herbicide-resistant corn in a certain ratio. The herbicide-resistant corn serves as a shelter, so that the selective pressure of target insects is reduced, and the generation of resistance is delayed.

Disclosure of Invention

In a first aspect, the present application provides a nucleic acid molecule comprising:

i) 1, nucleotide 367 and/or 634, and/or nucleotide 5904 and 6232, or a fragment or variant thereof or a complementary sequence thereof;

ii) the sequence which is indicated by nucleotides 1 to 634 and/or nucleotides 5904-6232 of SEQ ID NO.1, or a fragment or variant thereof or the complement thereof;

iii) the sequence depicted at nucleotide nos. 367 and 634 and/or 5904 and 6763 of SEQ ID NO.1, or a fragment or variant thereof or a complementary sequence thereof;

iv) the sequence shown by nucleotides 1 to 634 and/or nucleotides 5904-6763 of SEQ ID NO.1, or a fragment or variant thereof or a complementary sequence thereof; or

v) the sequence indicated by nucleotide 367 and 6232 of SEQ ID NO.1, or a fragment or variant thereof or a complementary sequence thereof.

In one embodiment, the nucleic acid molecule provided herein comprises the sequence shown in SEQ ID No.1, or a fragment or variant thereof or the complement thereof.

In another embodiment, a nucleic acid molecule provided herein comprises the following expression cassette:

a first expression cassette for expressing a glufosinate-resistant gene, comprising the sequence shown as nucleotides 405-1945 of SEQ ID NO. 1;

and a second expression cassette for expressing a glyphosate-resistant gene comprising the sequence shown by nucleotide 2266-6203 of SEQ ID NO: 1.

In another embodiment, the nucleic acid molecule provided herein is obtained by introducing into the genome of maize:

a first expression cassette for expressing an anti-glufosinate gene comprising the sequence shown as nucleotides 405-1945 of SEQ ID NO: 1;

and a second expression cassette for expressing a glyphosate-resistant gene comprising the sequence shown by nucleotide 2266-6203 of SEQ ID NO: 1.

The nucleic acid molecules provided herein are present in a maize plant, seed, plant cell, progeny plant or plant part.

In a second aspect, the present application provides a probe for detecting a maize transformant, comprising the sequence shown at nucleotides 1 to 634 and/or nucleotides 5904-6763 of SEQ ID NO.1, or a fragment or variant thereof or the complement thereof.

In a third aspect, the present application also provides a primer pair for detecting a maize transformant, which is capable of specifically amplifying to generate a sequence comprising nucleotides 1 to 634 of SEQ ID NO.1 or nucleotides 5904-6763, or a fragment or variant thereof or a complementary sequence thereof.

In one embodiment, the primer pair is:

i) a primer pair which specifically recognizes a sequence shown by nucleotides 1 to 634 of SEQ ID NO. 1;

ii) a primer pair which specifically recognizes the sequence shown by nucleotides 5904-6763 of SEQ ID NO. 1;

iii) a forward primer which specifically recognizes a sequence comprising nucleotides 1 to 634 of SEQ ID NO.1 and a reverse primer which specifically recognizes a sequence comprising nucleotides 368 and 6231 of SEQ ID NO. 1;

iv) a forward primer specifically recognizing the sequence as represented by nucleotide 368-6231 of SEQ ID NO.1 and a reverse primer specifically recognizing the sequence as represented by nucleotide 5904-6763 of SEQ ID NO. 1; or

v) a primer pair which specifically recognizes a sequence comprising the sequences shown by nucleotides 1 to 634 of SEQ ID NO.1 and a primer pair which specifically recognizes a sequence comprising the sequences shown by nucleotides 5904-6763 of SEQ ID NO. 1.

In one embodiment, the primer pair provided herein is the nucleotide sequence shown in SEQ ID NO. 18 and SEQ ID NO. 19 or the complementary sequence thereof; and/or the nucleotide sequences shown in SEQ ID NO:20 and SEQ ID NO:21 or the complementary sequences thereof.

In a fourth aspect, the present application also provides a kit or microarray for detecting a maize transformant, comprising the probe of the second aspect and/or the primer pair of the third aspect.

In a fifth aspect, the present application provides a method for detecting a maize transformant, comprising detecting the presence or absence of the transformant in a test sample using: a probe according to the second aspect; the primer set according to the third aspect; the probe of the second aspect and the primer pair of the third aspect; or a kit or microarray of the fourth aspect.

In a sixth aspect, the present application also provides a method of breeding maize, the method comprising the steps of: 1) obtaining maize comprising the nucleic acid molecule of the first aspect; 2) subjecting the maize obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain progeny plants, seeds, plant cells, progeny plants or plant parts; and optionally, 3) carrying out herbicide glufosinate-ammonium and glyphosate resistance and borer resistance identification on the progeny plants obtained in the step 2), and detecting whether the transformants exist by using the method.

In a seventh aspect, the present application also provides corn plants, seeds, plant cells, progeny plants or plant parts, and the like, obtained by the above-described methods, and articles made from these corn plants, seeds, plant cells, progeny plants or plant parts, and the like, including food, feed, or industrial materials, and the like.

Drawings

FIG. 1 is a schematic structural view of vector pZHZH15032, in which:

T-Border (right) T-DNA right border sequence

NOS terminator of terminator nopaline synthase NOS

cP4 EPSPS encoding EPSPS glyphosate tolerant herbicide gene

Omega sequence derived from tobacco etch virus gene expression enhancing elements

ubiquitin promoter from maize

CaMV 35S promoter cauliflower mosaic virus 35S promoter

Bar anti-glufosinate gene sequence

CaMV 35S terminator cauliflower mosaic virus 35S terminator

T-Border (left) T-DNA left border sequence

Kanamycin (R) kanamycin resistance sequence

pBR322 ori pBR322 initiation region sequence

pBR322 bom pBR322 framework region sequence

pVS1 rep pVS1 replicon

pVS1 sta pVS1 transcriptional initiation region

bp base pair

FIG. 2 is a photograph of plants one week after treatment with the glufosinate herbicide "Bauda", wherein:

a is non-transgenic negative control wild type 249, glufosinate-ammonium is not sprayed, the plant grows normally, and no damage symptom exists;

b is non-transgenic negative control wild type 249, 250 ml/mu of 'Baotai' is sprayed, after one week, leaves are dry, green, withered and stagnated, and obvious phytotoxicity symptoms are shown;

c is ZZM032T 032 ₃ In generation, 250 ml/mu of 'guarantor' is sprayed, and plants grow normally after one week without any damage symptoms;

d is ZZM032T ₄ And in generation, 250 ml/mu of' conservation test is sprayed, and the plant grows normally after one week without any damage symptom.

FIG. 3 is a photograph of a plant treated for one week with glyphosate herbicide spray "Nondata", wherein:

a is non-transgenic negative control wild type culture 249, no agricultural product is sprayed, the plant grows normally, and no damage symptom is caused;

b is non-transgenic negative control wild type 249, 200 ml/mu of 'nongda' is sprayed, after one week, leaves are dry, green, withered and stagnated, and obvious phytotoxicity symptoms are shown;

c is ZZM032T 032 ₃ In generation, 200 ml/mu of 'nongda' is sprayed, the plant grows normally after one week, and no damage symptom exists;

d is ZZM032T ₄ And in generation, 200 ml/mu of 'nongda' is sprayed, and the plant grows normally after one week without any damage symptoms.

FIGS. 4A and 4B are the results of the copy number detection of ZZM032 Southern blot, in which:

FIG. 4A shows the hybridization of the genomic DNA of maize of transformant ZZM032 with the bar-specific probe molecule by digestion with HindIII, EcoRI and KpnI, respectively. The three enzyme cutting conditions respectively show a positive band, the band obtained by enzyme cutting with HindIII is 3.4kb, the band obtained by enzyme cutting with EcoRI is 9.2kb, the band obtained by enzyme cutting with KpnI is 1.5kb, and the results are all in line with the expectation, and the single copy insertion of the exogenous gene bar is shown, and the transformant is a single copy transformant.

FIG. 4B shows the hybridization of the transformant ZZM032 maize genomic DNA cleaved with HindIII, EcoRI and KpnI + AflIII respectively to the cp4 epsps specific probe molecule. A positive band is shown under three enzyme digestion conditions respectively, the band obtained by enzyme digestion of HindIII is 9.2kb, the band obtained by enzyme digestion of EcoRI is 6.4kb, and the band obtained by enzyme digestion of KpnI + AflIII is 3.8kb, which are all in line with expectations, and indicate that the exogenous gene cp4 epsps is inserted in a single copy way, and the transformant is a single copy transformant.

FIG. 5 shows the result of PCR detection specific to the transformant ZZM032, wherein:

a is a left boundary detection result;

b is a right boundary detection result;

wherein lanes 1-4 are: sterile water, 249DNA, ZZM032T ₃ DNA and ZZM032T ₄ DNA。

Detailed Description

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be understood in accordance with their ordinary usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards and other publications cited herein, and the like, are incorporated herein by reference in their entirety.

As used herein, "maize" is any maize plant and includes all plant varieties that can be bred with maize, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, and plant cells intact in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like.

It is well known to those skilled in the art that expression of foreign genes in plants has a positional effect, i.e., is influenced by the location of insertion into the chromosome, which may be due to a chromosomal structure or transcriptional regulatory elements near the integration site. Therefore, it is usually necessary to produce hundreds of different transformants and to screen out excellent transformants with the desired expression level and pattern of the foreign gene from these events for commercial production applications.

The excellent transformant can transfer the exogenous gene into germplasm with other genetic backgrounds by a conventional breeding method, namely sexual hybridization, and the progeny of the excellent transformant maintains the transgenic expression characteristics of the original transformant. The present application relates to a superior transformant ZZM032 selected from among a plurality of transformants.

In this application, "transformant ZZM 032" refers to a maize plant transgenic for the inbred line 249 of maize to obtain an exogenous gene insert (T-DNA insert) inserted between specific genomic sequences, wherein the exogenous gene insert comprises the following two genes: glufosinate-resistant genes and glyphosate-resistant genes. The transformant ZZM032 obtained by the application has the advantages that the inserted exogenous gene is positioned at a non-functional site of a corn genome, the expression of other genes of the plant is not influenced, and the transgenic corn plant can obtain the herbicide resistance and simultaneously maintain the good agronomic characters.

In a specific example, the T-DNA insert obtained after the transgene has the sequence shown by nucleotides 368 and 6224 of SEQ ID NO.1 and the random insertion of 7bp nucleotides, such as the sequence shown by nucleotides 6225 and 6231 of SEQ ID NO. 1. Transformant ZZM032 may refer to this transgenic process, may also refer to the T-DNA insert within the genome resulting from this process, or the combination of the T-DNA insert and flanking sequences, or may refer to a maize plant resulting from this transgenic process. Transformant ZZM032 may also refer to progeny plants derived from the above plants by vegetative propagation, sexual propagation, doubling or doubling, or a combination thereof.

In other embodiments, this event is also applicable to plants obtained by transforming other plant recipient varieties with the same foreign gene (the sequence indicated by nucleotide 368 nd and 6224 th of SEQ ID NO: 1) and inserting the T-DNA insert into the same genomic position. Suitable plants include monocotyledons such as rice, wheat, oats, barley, highland barley, millet, sorghum, sugarcane, and the like.

In the present application, inserts were obtained with nucleotides 1-367 of SEQ ID NO:1 as the left flank sequence and nucleotides 6232-6763 of SEQ ID NO:1 as the right flank sequence (nucleotides 368-6231). The flanking sequences are not limited to nucleotides 1-367 and 6232-6763 of SEQ ID NO 1, since the listed flanking sequences are only used to indicate the position of the T-DNA insert in the genome, i.e.the insertion point to the left of the T-DNA insert is located on chromosome 9 at 133,993,669 bp; the right insertion point of the T-DNA insert is located on chromosome 9 at 133,993,710 bp. Thus, the flanking sequences of the present application may be flanked by genomic sequences, i.e., the left flanking sequence may extend 133,993,669bp upstream and the right flanking sequence may extend 133,993,710bp downstream of chromosome 9.

Since the transformant ZZM032 produces a T-DNA insert inserted into a specific site in the genome, its insertion site is specific and can be used to detect whether the transformant ZZM032 is contained in a biological sample. In particular embodiments, any sequence comprising the junction site of the T-DNA insert of transformant ZZM032 with flanking sequences may be used to detect transformant ZZM032 of the present application, including but not limited to one or more of the following sequences or fragments thereof or variants thereof or complements thereof comprising an upstream insertion site (the junction site of the left flanking sequence with the T-DNA insert) and/or a downstream insertion site (the junction site of the right flanking sequence with the T-DNA insert): i) comprises the sequence shown as nucleotides 367 and 634 of SEQ ID NO. 1; ii) comprises the sequence shown as nucleotide 5904-6232 of SEQ ID NO. 1; iii) comprises the sequence shown as nucleotides 1 to 634 of SEQ ID NO. 1; iv) comprises the sequence shown as nucleotide 5904-6763 of SEQ ID NO. 1; v) comprises the sequence shown in SEQ ID NO. 1.

In specific examples, sequences that can be used to detect transformant ZZM032 of the present application are sequences comprising an upstream insertion site or a fragment or variant thereof or the complement thereof, such as the sequences shown by nucleotides 1-634 of SEQ ID NO:1, or sequences comprising a downstream insertion site, such as the sequences shown by nucleotides 5904-6763 of SEQ ID NO:1, or a combination of a sequence comprising an upstream insertion site and a sequence comprising a downstream insertion site.

In another example, sequences useful for detecting the transformant ZZM032 of the present application are a combination of the sequence comprising the upstream insertion site or fragment thereof or variant thereof or complement thereof and the sequence comprising the T-DNA insert or fragment thereof or variant thereof or complement thereof, e.g., the sequence shown by nucleotides 1-634 of SEQ ID NO:1 in combination with the sequence shown by nucleotides 368-6231 of SEQ ID NO: 1.

In another example, sequences useful for detecting the transformant ZZM032 of the present application are a combination of a sequence comprising a downstream insertion site or fragment thereof or a variant thereof or the complement thereof and a sequence comprising a T-DNA insert or a fragment thereof or a variant thereof or the complement thereof, e.g., a sequence comprising nucleotides 5904-6763 of SEQ ID NO:1 in combination with a sequence comprising nucleotides 368-6231 of SEQ ID NO: 1.

In another example, the sequence that can be used to detect the transformant ZZM032 of the present application comprises the sequence shown in SEQ ID No.1 or a fragment or variant thereof or the complement thereof.

Thus, primer pairs, probes, and combinations of primer pairs and probes that are capable of specifically detecting the junction site of the T-DNA insert of transformant ZZM032 with flanking sequences can all be used to detect transformant ZZM032 of the present application.

As used herein, "nucleotide sequence" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, nucleotide sequences are written from left to right in the 5 'to 3' direction.

In some embodiments, the present application also relates to fragments of nucleic acid sequences, which refer to portions of smaller fragments that are incomplete in the complete portion. For example, SEQ ID NO:1 comprises SEQ ID NO:1, at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, or at least about 50 nucleotides of the complete sequence or more.

In some embodiments, changes may be made to the nucleic acid sequences of the present application to make conservative amino acid substitutions. In certain embodiments, substitutions that do not alter the amino acid sequence of the nucleotide sequences of the present application can be made according to monocot codon preferences, e.g., codons encoding the same amino acid sequence can be substituted with monocot preferred codons without altering the amino acid sequence encoded by the nucleotide sequence. In some embodiments, the present application also relates to variants of the nucleic acid sequences. Generally, variants of a particular nucleic acid fragment will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more sequence identity, or a complement thereof, to the particular nucleotide sequence. Such variant sequences include one or more additions, deletions or substitutions of nucleic acid residues, which may result in the addition, removal or substitution of corresponding amino acid residues. Sequence identity is determined by sequence alignment programs known in the art, including hybridization techniques. Nucleotide sequence variants of the embodiments may differ from the sequences of the present application by as little as 1-15 nucleotides, as little as 1-10 (e.g., 6-10), as little as 5, as little as 4, 3, 2, or even 1 nucleotide.

As used herein, a "probe" is an isolated polynucleotide, complementary to a strand of a target polynucleotide, to which is attached a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent or enzyme.

In a specific embodiment, the DNA probe provided herein for detecting transformant ZZM032 comprises a DNA sequence comprising SEQ ID NO:1 or a complete complement thereof, which DNA probe hybridizes under stringent hybridization conditions to a nucleotide sequence comprising an upstream insertion site or a downstream insertion site and does not hybridize under stringent hybridization conditions to a nucleotide sequence not comprising an upstream insertion site or a downstream insertion site.

In specific examples, the probes provided herein comprise the sequences shown at nucleotides 1-634 or nucleotides 5904-6763 of SEQ ID NO.1, or fragments or variants thereof or complements thereof. As used herein, a "primer" is an isolated polynucleotide that anneals to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then stretches along the target DNA strand by means of, for example, a DNA polymerase. Primer pairs are directed to their target polynucleotide amplification use, for example, by Polymerase Chain Reaction (PCR) or other conventional nucleic acid amplification methods.

In a specific embodiment, the primer pair for detecting the transformant ZZM032 provided herein comprises a first DNA molecule and a second DNA molecule different from the first DNA molecule, wherein the first and second DNA molecules each comprise the nucleotide sequence set forth in SEQ ID NO:1 or the complete complement thereof, and wherein the first DNA molecule is present in SEQ ID NO:1 and the second DNA molecule are present in SEQ ID NO:1 which when used in conjunction with DNA from the transformant ZZM032 in an amplification reaction produces an amplicon which is useful for detecting the transformant ZZM032 DNA in a sample, and wherein the amplicon comprises the sequence shown at nucleotides 1-634 or nucleotides 5904-6763 of SEQ ID No.1, or a fragment or variant or complement thereof.

In a specific embodiment, the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising nucleotides 1 to 634 of SEQ ID NO. 1.

In a specific embodiment, the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising nucleotides 5904-6763 of SEQ ID NO. 1.

In specific embodiments, the primer pairs provided herein are: i) a primer pair which specifically recognizes a sequence shown by nucleotides 1 to 634 of SEQ ID NO. 1; and ii) a primer pair which specifically recognizes a sequence comprising nucleotides 5904-6763 of SEQ ID NO. 1; alternatively, the primer pair comprises: a forward primer which specifically recognizes a sequence consisting of nucleotides 1 to 634 of SEQ ID NO.1, and a reverse primer which specifically recognizes a sequence consisting of nucleotides 5904-6763 of SEQ ID NO. 1.

In another embodiment, the primer pairs provided herein are: i) a primer pair which specifically recognizes a sequence shown by nucleotides 1 to 634 of SEQ ID NO. 1; and ii) a primer pair which specifically recognizes a sequence comprising the sequence depicted by nucleotide 368 nd and 6231 of SEQ ID NO. 1; alternatively, the primer pair comprises: a forward primer which specifically recognizes a sequence represented by nucleotides 1 to 634 of SEQ ID NO.1, and a reverse primer which specifically recognizes a sequence represented by nucleotides 368-6231 of SEQ ID NO. 1.

In another embodiment, the primer pairs provided herein are: i) a primer pair which specifically recognizes the sequence shown by the nucleotide 368-position 6231 of SEQ ID NO. 1; and ii) a primer pair which specifically recognizes a sequence comprising the sequence shown by nucleotides 5904-6763 of SEQ ID NO. 1; alternatively, the primer pair comprises: a forward primer which specifically recognizes the sequence shown by the nucleotide 368-6231 of SEQ ID NO.1, and a reverse primer which specifically recognizes the sequence shown by the nucleotide 5904-6763 of SEQ ID NO. 1.

In another embodiment, the primer pairs provided herein are: i) a primer pair which specifically recognizes the sequence represented by nucleotides 1 to 634 of SEQ ID NO.1, ii) a primer pair which specifically recognizes the sequence represented by nucleotides 368 and 6231 of SEQ ID NO.1, and iii) a primer pair which specifically recognizes the sequence represented by nucleotides 5904 and 6763 of SEQ ID NO. 1.

In another embodiment, the primer pair provided herein is a primer pair that specifically recognizes a sequence comprising SEQ ID NO. 1.

In a specific example, the primer pair is nucleotide sequences shown as SEQ ID NO. 18 and SEQ ID NO. 19 or complementary sequences thereof; or the nucleotide sequences shown in SEQ ID NO. 20 and SEQ ID NO. 21 or the complementary sequences thereof.

Methods of designing and using primers and probes are well known in the art, for example, in Joseph Sambrook, Molecular Cloning: a Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) and Current Protocols in Molecular Biology, Wiley-Blackwell.

As used herein, "kit" or "microarray" refers to a set of reagents or chips for the purpose of identification and/or detection of corn transformant ZZM032 in a biological sample. For the purpose of quality control (e.g. purity of seed lots), detection of transformant ZZM032 in or comprising plant material or material derived from plant material, such as but not limited to food or feed products, kits or chips may be used, and the components thereof may be specifically adjusted.

In particular embodiments, a kit or probe provided herein includes any one of the probes or any one of the primer pairs provided herein. In another specific embodiment, a kit or probe provided herein comprises any one of the probes provided herein or a combination of any one of the primer pairs.

In addition, transgenic corn plants, progeny, seeds, plant cells or plant parts, and preparations thereof, including but not limited to food, feed, or industrial materials, are provided. The plants, progeny, seeds, plant cells, plant parts, and preparations thereof all comprise a nucleic acid molecule sequence that is detectable as a site of engagement of the T-DNA insert provided herein with the flanking sequences.

Further, the present application also provides a method of breeding maize comprising the steps of: 1) obtaining maize comprising a nucleic acid molecule sequence comprising the junction site of the T-DNA insert provided herein with flanking sequences; 2) subjecting the maize obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain progeny plants, seeds, plant cells, progeny plants or plant parts; and optionally step 3), identifying the corn plants obtained in step 2) for resistance to herbicides glufosinate and glyphosate and resistance to borers, and detecting the presence or absence of the transformant ZZM032 therein by using the probe, primer pair, kit or array provided herein.

In addition, the present application provides methods of controlling weeds in a field.

In a specific embodiment, a method of controlling weeds in a field provided herein comprises planting a corn plant comprising transformant ZZM032 in a field and applying an effective dose of glyphosate and glufosinate herbicide in the field capable of controlling weeds without damaging the transgenic corn plant comprising transformant ZZM 032.

The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application. Modifications or substitutions to methods, steps or conditions disclosed herein may be made without departing from the spirit and scope of the present application.

Unless otherwise indicated, the examples are carried out according to conventional experimental conditions, such as the Molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory Manual,2001), or according to the conditions suggested by the manufacturer's instructions.

Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

The maize variety materials related to the following examples are all provided by the Chinese seed group company ltd, wherein the maize inbred line 249 is the female parent of the great wall 799 maize variety, maize germplasm resources introduced abroad are used as materials, and the maize inbred line materials are obtained by inbreeding separation and strict selection through a pedigree method and breeding in 1996 after 10 generations.

Example 1 vector construction

1. Synthesis of herbicide-resistant exogenous gene

The bar gene sequence refers to a sequence shown in Genebank accession number X17220.1, and a glufosinate-resistant herbicide gene shown in bases 613 to 1164 of SEQ ID NO 1 is synthesized in an artificial synthesis mode.

Entrusted Nanjing King Shirui Biotechnology Ltd optimizes and synthesizes epsps gene according to rice codon preference, as shown in base 4339 to 5931 of SEQ ID NO:1, and adds a segment of expression enhancement element omega sequence of 65 nucleotides at 5' end, as shown in base 4274 to 4338 of SEQ ID NO: 1.

2. Vector construction

The existing intermediate vector pZZ001142 containing nos terminator (XhoI restriction site at 5 'end and PmeI restriction site at 3' end) is subjected to single enzyme digestion by using restriction enzyme XhoI, a skeleton vector fragment is recovered, and the generated sticky end is filled with T4 DNA polymerase.

The existing intermediate vector pZZ00002 containing ubiquitin gene promoter-epsps-CaMV 35S terminator fragment (the 5 'end of ubiquitin gene promoter has PmeI and HindIII restriction sites, and the 3' end of epsps has SacI restriction site) is subjected to double enzyme digestion by restriction enzymes PmeI and SacI, the ubiquitin gene promoter-epsps fragment is recovered, and the sticky end generated is filled by T4 DNA polymerase.

The backbone vector fragment containing the nos terminator and the ubiquitin gene promoter-epsps fragment were ligated by T4 DNA ligase to obtain a vector containing the ubiquitin gene promoter-epsps-nos terminator fragment, which was named pZZ 001199.

Carrying out double enzyme digestion by using restriction enzymes HindIII and PmeI on the existing intermediate vector pZZ00015 containing a CaMV 35S promoter-bar-CaMV 35S terminator and a ubiquitin gene promoter-gfp-nos terminator fragment (the 5 'end of the ubiquitin gene promoter is provided with a HindIII enzyme cutting site, and the 3' end of the nos terminator is provided with a PmeI enzyme cutting site), cutting the ubiquitin gene promoter-gfp-nos terminator fragment, and recovering to obtain the skeleton vector fragment with the CaMV 35S promoter-bar-CaMV 35S terminator.

pZZ001199 was treated with restriction enzymes HindIII and PmeI, and the ubiquitin gene promoter-epsps-nos terminator fragment was recovered. T4 DNA ligase is connected with ubiquitin gene promoter-epsps-nos terminator fragment and vector fragment containing CaMV 35S promoter-bar-CaMV 35S terminator skeleton, so as to obtain a transformation vector, which is named as pZHZHZH 15032. The physical map is shown in figure 1.

Example 2 obtaining of transgenic maize

Transgenic maize is obtained using agrobacterium-mediated genetic transformation methods.

The plasmid DNA of the vector pZHZH15032 was transformed into Agrobacterium EHA105 by electroporation and identified for use.

Maize was then transformed with EHA105, which identified suitable agrobacterium. In brief, young embryos with the length of about 1.5mm are taken for transformation after selfing of a maize inbred line 249. Collecting young embryos of about 200 ears into a batch, placing the batch in an EP tube, sucking out the suspension, adding an agrobacterium liquid containing 200 mu M acetosyringone, co-culturing for 5 minutes, transferring the young embryos to a co-culture medium, and culturing for three days in the dark. Placing the dark-cultured immature embryos on a rest culture medium, placing the immature embryos on a selective culture medium containing 5mg/L bialaphos after callus grows out, screening and culturing, and carrying out subculture once every two weeks. When resistant callus grows out, selecting out embryogenic callus with good state, transferring to differentiation culture medium under the culture condition of 26 deg.C, 3000Lux light intensity every day, and illuminating for 16 hr, and generating regeneration plantlet after two weeks. The regenerated plantlet is transferred to a rooting culture medium, and after the plantlet grows secondary roots, the plantlet is transplanted to a small pot mixed with nutrient soil and vermiculite (1: 3) (the specific method and the culture medium refer to the efficient transgenic method of the corn backbone inbred line disclosed in the Chinese invention patent application CN104745622A of the applicant).

The obtained transformed seedlings are subjected to transgenic positive detection according to the following steps, and transgenic positive plants are selected.

(1) DNA extraction

The corn genomic DNA was extracted using DNAsecure Plant Kit novel Plant genomic DNA extraction Kit (centrifugal column type) from Tiangen Biochemical technology.

(2)PCR

The following reagents were thawed from a-20 ℃ freezer: 10-fold PCR buffer (Takara), deoxynucleotide mix (10mM, Sigma), forward primer SEQ ID NO: 2: 5'-CAGCACAGGTTAAGTCTG-3', reverse primer SEQ ID NO: 3: 5'-GTCTGTCTCAACGGTAAG-3', and maize leaf DNA templates. After thawing all reagents, centrifuge for several seconds and place on ice for use. And preparing a mixed solution of a PCR reaction system, uniformly mixing, and centrifuging for several seconds. PCR reaction (20. mu.L): mu.L of 10-fold PCR buffer (Takara), 0.5. mu.L of deoxynucleotide mix (10mM, Sigma), 0.8. mu.L of forward and reverse primer mix (5. mu.M), 0.2. mu. L r-Taq (5U, Takara), 1. mu.L of maize leaf DNA template, and the balance dd H ₂ And O. The mixture was dispensed into 200. mu.L PCR tubes, and 1. mu.L template DNA was added and labeled separately for each sample. Placing the PCR reaction tube into Thermo 9700 typeAnd the PCR amplification instrument selects a preset PCR amplification program and starts to operate the reaction. The PCR reaction program is: pre-denaturation at 94 ℃ for 2 min; 30 cycles of: denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds; final extension at 72 ℃ for 5 min.

(3) Agarose gel electrophoresis detection

After the PCR is finished, 5 μ L of PCR product is taken for agarose gel electrophoresis detection. A1.5% agarose gel was prepared, stained in Ethidium Bromide (EB) for 10 minutes after electrophoresis at 150V for 25 minutes, and photographed in an ultraviolet gel imaging system.

(4) Determination of the result

The material capable of amplifying the 140bp band is a transgenic positive plant, and the material incapable of amplifying the band is a transgenic negative plant.

Transplanting the transgenic positive plant into a large flowerpot to obtain T ₀ And (5) plant generation.

Example 3 resistance identification of transgenic maize

T ₀ Selfing the plant, the obtained seed is T ₁ And (5) seed generation. Will T ₁ Seeding the seeds in a greenhouse to obtain T ₁ And (5) plant generation. Repeating the above process until T is obtained ₄ And (5) seed generation.

For T ₁ To T ₃ And carrying out transgenic positive detection, herbicide resistance analysis and identification and agronomic character analysis on the generation plants. Selecting out plants with positive transgenes, herbicide resistance and excellent agronomic characters from each generation of plants, and entering next generation screening.

1. Positive detection

The detection method and procedure were as described in example 2.

2. Herbicide resistance character identification

And (3) sowing the selfed seeds of the positive plants detected in the step (1) into a greenhouse, carrying out herbicide resistance identification on plants in a 6-8 leaf stage, and removing the plants which do not tolerate the herbicide.

(1) Glufosinate herbicide tolerance identification

The glufosinate herbicide 'Baozida' (Basta) used for spraying is produced by Bayer crop science (China) limited company, and the active ingredient is 18% glufosinate-ammonium soluble agent. The recommended dosage of the herbicide is 200-300 ml/mu, and the herbicide is sprayed by adopting the dosage of 250 ml/mu in the recommended concentration. Herbicide tolerance performance was observed and recorded after one week. The corn plants with the tolerance to glufosinate-ammonium grow normally and have no damage symptom; maize plants sensitive to glufosinate exhibit significant phytotoxicity symptoms including growth arrest, chlorosis, blight, deformity, and the like, until the entire plant dies.

At T ₁ 、T ₂ And T ₃ In the generation plant population, the Chi-square test was performed according to the following formula, based on the actual segregation ratio for which glufosinate resistance was observed and the expected segregation ratio calculated according to mendelian's law of inheritance (table 1): chi shape ² ＝Σ[(|o–e|–0.5) ² /e](ii) a Wherein, o is the observed value of the number of positive plants or the number of negative plants, e is the expected value of the number of positive plants or the number of negative plants, and 0.5 is a Yates analysis correction factor when the degree of freedom is 1.

TABLE 1 expected segregation ratio for each generation of transformant ZZM032

*: in the case of single site single copy insertion, the expected segregation ratio calculated according to Mendelian's Law of inheritance.

In table 2, the "observed values" are the actual number of positive plants and the actual number of negative plants observed after glufosinate-ammonium spraying; the "expected value" is the number of theoretical transgenic positive plants and the number of transgenic negative plants calculated according to the Mendelian's Law of inheritance in accordance with Table 1; 'chi' for treating rheumatism ² "is a Chi square value calculated according to a Chi square test formula; 'X' type ² _0.05,1 "is the degree of freedom of 0.05 at significance level α and 1 degree of freedom ² Values obtained from the boundary value table; "probability" is x ² And chi ² _0.05,1 The result of comparison between the two is when x ² <χ ² _0.05,1 When it is, P>0.05, indicating no significant difference between observed and expected values. χ in Table 2 ² Test analysis meterMing at T ₁ -T ₃ No significant difference (P) was observed between the observed and expected genetic segregation ratios in the investigated generations>0.05), indicating ZZM032 stably inherited according to Mendelian inheritance rule between generations and at T ₂ The generation is homozygous.

TABLE 2 isolation analysis of transformant ZZM 032-X for each generation ² Examination of

(2) Glyphosate herbicide tolerance identification

The glyphosate herbicide "nongda" (Roundup) used for spraying is produced by Monsanto company, and the active ingredient is 41% isopropylamine salt. The recommended dosage of the herbicide in the corn field is 150-. The corn plants with the glyphosate tolerance grow normally without any damage symptom; corn plants sensitive to glyphosate show significant phytotoxicity symptoms including growth inhibition, chlorosis, blight, deformity, and the like, until the entire plant dies.

3. Maize transformant ZZM032

Through the above process, the maize transformant ZZM032 was finally screened.

The results are shown in FIGS. 2 and 3. FIG. 2 is a photograph of a plant one week after "Protect Up" for a glufosinate herbicide, wherein: a is non-transgenic negative control wild type 249, glufosinate-ammonium is not sprayed, the plant grows normally, and no damage symptom is caused; b is non-transgenic negative control wild type 249, 250 ml/mu of 'conservation test reach' is sprayed, after one week, leaves are dry, green, withered spots and growth retardation, and obvious phytotoxicity symptoms are shown; c is ZZM032T 032 ₃ In generation, 250 ml/mu of 'guarantor' is sprayed, and plants grow normally after one week without any damage symptoms;d is ZZM032T ₄ And in generation, 250 ml/mu of' conservation test is sprayed, and the plant grows normally after one week without any damage symptom. FIG. 3 is a photograph of a plant one week after spraying glyphosate herbicide "Nondata", wherein: a is non-transgenic negative control wild type Xiang 249, no 'nongda' is sprayed, the plant grows normally, and no damage symptom exists; b is non-transgenic negative control wild type 249, 200 ml/mu of Nondata is sprayed, after one week, leaves are dry, green, withered spots and growth retardation, and obvious phytotoxicity symptoms are shown; c is ZZM032T 032 ₃ In generation, 200 ml/mu of 'nongda' is sprayed, the plant grows normally after one week, and no damage symptom exists; d is ZZM032T ₄ And in generation, 200 ml/mu of 'nongda' is sprayed, and the plant grows normally after one week without any damage symptoms.

EXAMPLE 4 identification of transformant ZZM032 by Southern blotting

1. Preparation of Probe

(1) Preparation of the Bar Probe

The probe was prepared using pZHZH15032 plasmid DNA as a template. Probes for detecting bar were synthesized using csp73(CAGTTCCCGTGCTTGAAG, SEQ ID NO:4) and csp74(CACCATCGTCAACCACTAC, SEQ ID NO:5) as primers and using Roche PCR digoxin probe synthesis kit (cat # 11636090910), and the size of the probe was 408bp (the probe sequence is identical to the nucleotide sequence shown in SEQ ID NO.1, position 692-1099). The amplification system comprises: 5. mu.L (50pg) of DNA template, 0.5. mu.L of each primer, 5. mu.L of PCR DIG mix, 0.75. mu.L of DNA polymerase, 5. mu.L of PCR buffer (10-fold), ddH ₂ O33.25. mu.L. The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; 35 cycles: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 45 seconds; final extension at 72 ℃ for 7 min. The effect of the marker amplification was detected using a 1% agarose gel. The specifically amplified amplification product, i.e.the probe for detecting bar, was stored at-20 ℃.

(2) Preparation of cp4 epsps Probe

Using the same method as described in (1), a specific probe for the cp4 epsps gene was prepared using the primer pairs csp91 and csp92 (sequences TCAATAGCAGCAACCTCTC, SEQ ID NO:6 and ATAGTCCTCACGCCATCA, SEQ ID NO:7, respectively) which was 856bp in length (the probe sequence is identical to the nucleotide sequence shown in positions 4385-5240 of SEQ ID NO: 1).

DNA extraction

Extracting total DNA of leaf genome of transgenic corn, drying the obtained DNA precipitate, dissolving in deionized water, and measuring the concentration for later use.

3. Enzyme digestion, electrophoresis, film transfer and development

When using the bar probe, the maize genomic DNA is cleaved separately with HindIII, EcoRI and KpnI.

When the cp4 epsps probe was used, the corn genomic DNA was cleaved with HindIII, EcoRI and KpnI + AflIII combinations, respectively.

Using 200. mu.L of a restriction enzyme system containing 20. mu.g of maize genomic DNA, 20. mu.L of restriction enzyme, 20. mu.L of 10-fold buffer, add ddH ₂ The volume of O is up to 200. mu.L. Carrying out electrophoresis detection on 10 mu L of the obtained product after digestion for 16 hours, and detecting whether the digestion effect is complete.

Enzyme digestion product plus ddH ₂ O was replenished to 400. mu.L, 1/10 volumes of 3M sodium acetate solution (pH5.2) were added, 4. mu.L of TaKaRa Dr.GenTLE Precipitation Carrier was added, 2.5 volumes of absolute ethanol were added, well mixed, and centrifuged at 12,000rpm at 4 ℃ for 15 minutes. The precipitate was washed with 50. mu.LddH ₂ O dissolved, and 10. mu.L of 6-fold loading buffer was added.

The DNA was run through a 0.8% gel at 20V for 16 h. Excess lanes and wells were cut off and the remaining gel was treated with denaturing solution 2 times for 15 min each time and gently shaken on a shaker. The mixture was treated with the neutralization buffer 2 times for 15 minutes each time and gently shaken on a shaker. And cleaning once with ultrapure water. The 20-fold SSC treatment is carried out for 10 minutes, and the membrane transfer is carried out for 4 hours or more using a Whatman system.

After the transfer of the membrane was complete, the membrane was placed on Whatman 3MM filter paper soaked with 10 times SSC and cross-linked for 3-5 minutes using a UV cross-linker. By ddH ₂ And O, simply washing the membrane and drying in the air. The hybridization and development were carried out according to the manual of Roche digoxin assay kit I (cat # 11745832910) or Roche digoxin assay kit II (cat # 11585614910).

4. Analysis of results

FIG. 4A shows the hybridization of the genomic DNA of maize of transformant ZZM032 with the bar-specific probe molecule by digestion with HindIII, EcoRI and KpnI, respectively. The three enzyme cutting conditions respectively show a positive band, the band obtained by enzyme cutting with HindIII is 3.4kb, the band obtained by enzyme cutting with EcoRI is 9.2kb, and the band obtained by enzyme cutting with KpnI is 1.5kb, which are consistent with expectations, and show that the exogenous gene bar is inserted in a single copy way, and the transformant is a single copy transformant.

FIG. 4B shows the hybridization of the transformant ZZM032 maize genomic DNA cleaved with HindIII, EcoRI and KpnI + AflIII respectively to the cp4 epsps specific probe molecule. The three digestion conditions respectively show a positive band, the band obtained by HindIII digestion is 9.2kb, the band obtained by EcoRI digestion is 6.4kb, and the band obtained by KpnI + AflIII digestion is 3.8kb, which are consistent with the expectation, and the single copy insertion of the exogenous gene cp4 epsps is shown, and the transformant is a single copy transformant.

Example 5 sequence analysis of transformant ZZM032

In transgenic operations, a large number of genetic transformations are generally performed using the same transformation vector, and few excellent transformants are selected from among the obtained many transformants. Therefore, the detection of the vector, expression element, foreign gene, etc. in the inserted foreign sequence can only prove that the test sample contains the transgenic component, and different transformants cannot be distinguished. The different transformants are characterized by a combination of flanking sequences at their insertion site and inserted foreign sequences. To this end, flanking sequences of maize transformants were isolated and identified.

1. Left flank sequence analysis

Taking the leaf of the transformant to be tested to extract the total DNA (T) ₂ Generation or T ₃ Strong transgenic plants grow for generations), and a linker PCR (adapter-PCR) method is utilized to amplify, clone and sequence the flanking sequence of the exogenous gene inserted into the corn genome to obtain a sequence result.

The method comprises the following specific steps:

(1) artificially synthesizing primers required by joint PCR, and diluting for later use; the primer sequences are shown in Table 3.

TABLE 3 primer sequences

(2) Preparing high-quality corn genome DNA for later use, and diluting the high-quality corn genome DNA for later use;

(3) by ddH ₂ O, respectively diluting the joint primers AD-L and AD-S to 100 mu mol/L, mixing in equal volume, carrying out water bath denaturation at 94 ℃ for 4min, and naturally cooling to room temperature to obtain a joint of 50 mu mol/L;

(4) the corn genome DNA is cut by enzyme, and the cutting system is as follows:

the enzyme was cleaved at 37 ℃ for 3 h.

(5) The connecting joint comprises the following connecting systems:

ligation was performed overnight at 16 ℃.

(6) Taking the genome DNA enzyme digestion-joint connection product in the step (5) as a template of a first round of PCR reaction, wherein the reaction system is as follows:

(7) the reaction procedure is as follows: 94 ℃, 5 min; (94 ℃, 30 sec; 72 ℃, 3 min). times.7 cycles; (94 ℃, 30 sec; 67 ℃, 3 min). times.32 cycles; 7min at 67 ℃; 25 ℃ for 10 min.

(8) And (3) performing second round PCR amplification by using the first round PCR product (diluted by 40 times in the mixed solution) as a template, wherein the reaction system is as follows:

(9) the reaction procedure is as follows: 94 ℃ for 5 min; (94 ℃, 30 sec; 72 ℃, 3 min). times.5 cycles; (94 ℃, 30 sec; 67 ℃, 3 min). times.20 cycles; 7min at 67 ℃; 25 ℃ for 10 min.

(10) Taking the product of the second round of PCR to carry out electrophoresis detection in 1% (w/v)1 xTAE agarose gel, and recovering a DNA fragment between 300bp and 2 kb;

(11) the recovered fragments were ligated to T-vector and ligated overnight at 16 ℃;

(12) converting the ligation product of step (11);

(13) amplifying the transformation product in the step (12) by using M13F and M13R primers, selecting a positive clone shake culture liquid, and extracting Plasmid DNA by using a TIAnprep Rapid Mini Plasmid Kit (centrifugal column type); sequencing primers are M13F and M13R; M13F: 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO:14), M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO: 15);

(14) sequencing the plasmid DNA of step (13) with sequencing primers using

Plasmid DNA was sequenced by PCR using Terminator v3.1 Cycle Sequencing Kit.

(15) And (3) purifying the PCR product in the step (14) by using NaAc and absolute ethyl alcohol and denaturing formamide.

(16) And (5) starting sequencing the purified and denatured PCR product in the step (15) by using an ABI DNA sequencer 3730 and reading a sequencing result.

(17) Sequencing results a homology search was performed with the maize genomic sequence in the Plant GDB database using the BLASTN tool, with the best match results being the chromosome number and base pair position number of the insertion site, typically 90-100% sequence identity.

(18) The left sequence 634bp of the inserted T-DNA is detected by the embodiment, and is shown as the sequence SEQ ID NO:1, nucleotides 1-634. The left insertion point of the transformant of the present application was determined to be located on chromosome 9, 133,993,669bp, by analysis and comparison with reference to the whole genome sequence of maize B73 (http:// www.plantgdb.org/ZmGDB/cgi-bin/blastGDB. pl). Obtaining a sequence of the foreign gene at the left border 634bp length of the integration site of the maize genome, comprising the maize genome sequence of 1-367 bp together and the vector left border sequence of 368-634 bp together.

2. Right flank sequence analysis

Taking the leaf of the transformant to be tested to extract the total DNA (T) ₂ Generation or T ₃ Robust transgenic plants were grown for generation), primers were designed using the left flank insertion site database whole genome sequence:

right flank vector primer:

csp1138：5’-TGAACTGTCGGATACCAAGGCGG-3’(SEQ ID NO:16)；

right flanking genomic primer:

csp5891：5’-GTTGGGCGTCGTTTGTCAT-3’(SEQ ID NO:17)。

the common PCR method is used for amplifying, cloning and sequencing the flanking sequence of the exogenous gene inserted into the corn genome to obtain a sequence result which is shown as a sequence SEQ ID NO:1 nucleotide 5904-6763. The right insertion point of the transformant of the present application was determined to be located on chromosome 9, 133,993,710bp, by analysis and comparison with reference to the maize B73 whole genome sequence (http:// www.plantgdb.org/ZmGDB/cgi-bin/blastGDB. pl).

3. Size of the insert and Effect on the endogenous genome of maize

The vector size of the exogenous gene single copy insertion sequence including the left and right border sequences is 5937 bp.

Through a segmented PCR amplification exogenous DNA sequencing method, the fact that 38bp of a T-DNA insert in a transformant is deleted relative to the left end of an expression vector, 42bp of the T-DNA insert is deleted at the right end of the expression vector, and 7bp of the T-DNA insert is randomly inserted is determined. The actual size of the insert was 5864bp (368-FIG. 6231 of SEQ ID NO: 1), including 5857bp of the T-DNA insert (368-FIG. 6224 of SEQ ID NO: 1), and 7bp of the random insert on the right (6225-FIG. 6231 of SEQ ID NO: 1).

The amplification system comprises: 2. mu.L (200ng) of DNA template, 0.5. mu.L each of primers (see Table 4 for primer sequences), 0.5. mu.L of DNA polymerase, 2. mu.L of PCR buffer (10-fold), ddH ₂ O14.5. mu.L. The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; 35 cycles of: denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 3 minutes; final extension at 72 ℃ for 7 min. The effect of the marker amplification was detected using a 1% agarose gel. Recovering the specifically amplified amplification product, i.e., the target fragment.

TABLE 4 flanking sequence PCR amplification primer information

The deleted nucleotides are located at the border of the non-coding region. The deletion of the gene does not influence the integrity of the herbicide-resistant genes bar and cp4 epsps. DNA sequence analysis and comparison show that the actually inserted nucleotide sequence of the transformant has a base mutation (T is changed into G at 4824 of SEQ ID NO: 1) to change the codon from CUU to CUG, and the mutation is a synonymous codon, so that the normal expression of the amino acid is not influenced.

In addition, the maize genome at the insertion site is a repetitive sequence, and nucleotide deletions do not disrupt any known maize endogenous functional genes.

Example 6 detection method of transformant ZZM032

1. Left flank DNA sequence detection:

a pair of primers FW-csp7743 and RV-Bar-22 are designed by utilizing the left flank genome sequence of the corn transformant and the sequence of Bar in the exogenous fragment, and a qualitative PCR identification method of the transformant product is established.

Primers designed according to the Left Border (LB) T-DNA 5' end of the integration site of the exogenous DNA fragment of the ZZM032 transformant are as follows:

FW-csp7743: 5'-TCCTACTACTGTTTACCATGCTTG-3' (SEQ ID NO:18, maize genomic region);

RV-Bar-22: 5'-CCTGCCCGTCACCGAGATTTGA-3' (SEQ ID NO:19, Bar region).

The specific primers are used for amplifying DNA fragments at 50-60 ℃ by using temperature gradient PCR to determine the optimal annealing temperature. Results demonstrate that the optimal amplification temperature is 58 ℃; the PCR procedure was 95 ℃ for 5min, (95 ℃ 30s, 58 ℃ 30s, 72 ℃ 1min)35 cycles, 72 ℃ for 7 min.

To test the characteristics of the transformants specifically amplified with the above primers (FW-csp 7743; RV-Bar-22), maize DNA from various sources was used for PCR amplification.

PCR reaction conditions and proceduresThe sequence was 95 ℃ for 5min, 35 cycles below: 95 ℃ for 30s, 58 ℃ for 30s, and 72 ℃ for 1 min; 7min at 72 ℃. The results showed that only the present transformant DNA could have a positive result, and the negative control maize was a negative result, as shown in FIG. 5A. Wherein lanes 1-4 are sterile water, 249DNA, ZZM032T ₃ DNA and ZZM032T ₄ DNA。ZZM032 T ₃ And T ₄ Lanes 3 and 4 of the genomic DNA show clearly visible bands, the size of the DNA fragment of 634bp is consistent with the expected size, and the sequencing of the DNA fragment clone is also consistent with the expected size.

2. Right flanking DNA sequence detection:

a pair of primers (SEQ ID NO:20: FW-csp1138 and SEQ ID NO:21: RV-csp5891) is designed by utilizing the cp4 epsps sequence in the exogenous fragment and the right flank genome sequence of the corn transformant, and a qualitative PCR identification method of the transformant is established.

Primers designed according to Right Border (RB) T-DNA 5' end of the integration site of the exogenous DNA fragment of the ZZM032 transformant are as follows:

FW-csp1138: 5'-TGAACTGTCGGATACCAAGGCGG-3' (SEQ ID NO:20, cp4 epsps region),

RV-csp5891: 5'-GTTGGGCGTCGTTTGTCAT-3' (SEQ ID NO:21, maize genomic region).

The specific primers are used for amplifying DNA fragments at 50-60 ℃ by using temperature gradient PCR to determine the optimal annealing temperature. Results demonstrate that the optimal amplification temperature is 58 ℃; the PCR reaction program is 95 ℃ for 5 min; 35 cycles of: 30s at 95 ℃, 30s at 58 ℃ and 1min at 72 ℃; 7min at 72 ℃.

To test the characteristics of the transformants specifically amplified by the above primers (FW-csp 1138; RV-csp5891), maize DNA from various sources was used for PCR amplification. The PCR reaction conditions and procedures were 95 ℃ for 5 min; 35 cycles of: circulating at 95 deg.C for 30s, 58 deg.C for 30s, and 72 deg.C for 1 min; 7min at 72 ℃. The results showed that only the present transformant DNA gave a positive result, and the negative control maize variety gave a negative result, as shown in B in FIG. 5. Wherein lanes 1-4 are sterile water, 249DNA, ZZM032T ₃ DNA and ZZM032T ₄ DNA; only ZZM032T ₃ And T ₄ The genome DNA lane has a clearly visible band, and the size of the DNA fragment is 860bpIn agreement, the results obtained by sequencing the DNA fragment clones were also in agreement with expectations.

The foregoing is only a preferred embodiment of the present application, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present application.

Reference to the literature

Joseph Sambrook，Molecular Cloning:A Laboratory Manual，Third Edition，Cold Spring Harbor Laboratory Press(2001).

Current Protocols in Molecular Biology,Wiley-Blackwell.

Sambrook J&Russell DW,Molecular cloning:a laboratory manual,2001.

Sequence listing

<110> China seed group Co., Ltd

<120> herbicide-resistant corn transformant and method for producing same

<130> 19C13699CN

<160> 35

<170> SIPOSequenceListing 1.0

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<211> 6763

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tcctactact gtttaccatg cttgcaatgc ttcttttgta cttttatgca agaatgcaaa 60

agtagttgct aggaagttgg gatccaaata caagggagac aaaacttgca tatgggttcc 120

aaaaactgtt gtgactaacc ttgtagaacc caacaagagt tgggtaccta aaacccaagt 180

ttaaatttct tgcaggttta tgcatccggg gactcaagct ggattatcga cagcggatgc 240

acaaaccata tgacggggga gaagaagatg ttcacctcct acgtcaagaa caaggattcc 300

caggatacga tcatctttgg agatgggaat caaggcaagg tcaaaggttt ggacaagata 360

gccatcagac aacttaataa cacattgcgg acgtttttaa tgtactgaat taacgccgaa 420

ttaattcggg ggatctggat tttagtactg gattttggtt ttaggaatta gaaattttat 480

tgatagaagt attttacaaa tacaaataca tactaagggt ttcttatatg ctcaacacat 540

gagcgaaacc ctataggaac cctaattccc ttatctggga actactcaca cattattatg 600

gagaaactcg agtcaaatct cggtgacggg caggaccgga cggggcggta ccggcaggct 660

gaagtccagc tgccagaaac ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg 720

cagcatgccg cggggggcat atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg 780

cagcccgatg acagcgacca cgctcttgaa gccctgtgcc tccagggact tcagcaggtg 840

ggtgtagagc gtggagccca gtcccgtccg ctggtggcgg ggggagacgt acacggtcga 900

ctcggccgtc cagtcgtagg cgttgcgtgc cttccagggg cccgcgtagg cgatgccggc 960

gacctcgccg tccacctcgg cgacgagcca gggatagcgc tcccgcagac ggacgaggtc 1020

gtccgtccac tcctgcggtt cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat 1080

gtagtggttg acgatggtgc agaccgccgg catgtccgcc tcggtggcac ggcggatgtc 1140

ggccgggcgt cgttctgggc tcatggtaga ctcgagagag atagatttgt agagagagac 1200

tggtgatttc agcgtgtcct ctccaaatga aatgaacttc cttatataga ggaagggtct 1260

tgcgaaggat agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc 1320

acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg 1380

ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg 1440

caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag 1500

atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca 1560

atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg 1620

tgctccacca tgttcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt 1680

ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc 1740

ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac cttccttttc 1800

tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga 1860

tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg tatctttgat 1920

attcttggag tagacgagag tgtcgtgctc caccatgttg gcaagctgct ctagccaata 1980

cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt 2040

cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag 2100

gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga 2160

taacaatttc acacaggaaa cagctatgac atgattacga attcgagctc ggtacccggg 2220

gatcctctag agtcgacctg caggcatgca agcttgcatg cctgcagtgc agcgtgaccc 2280

ggtcgtgccc ctctctagag ataatgagca ttgcatgtct aagttataaa aaattaccac 2340

atattttttt tgtcacactt gtttgaagtg cagtttatct atctttatac atatatttaa 2400

actttactct acgaataata taatctatag tactacaata atatcagtgt tttagagaat 2460

catataaatg aacagttaga catggtctaa aggacaattg agtattttga caacaggact 2520

ctacagtttt atctttttag tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc 2580

tatataatac ttcatccatt ttattagtac atccatttag ggtttagggt taatggtttt 2640

tatagactaa tttttttagt acatctattt tattctattt tagcctctaa attaagaaaa 2700

ctaaaactct attttagttt ttttatttaa taatttagat ataaaataga ataaaataaa 2760

gtgactaaaa attaaacaaa taccctttaa gaaattaaaa aaactaagga aacatttttc 2820

ttgtttcgag tagataatgc cagcctgtta aacgccgtcg acgagtctaa cggacaccaa 2880

ccagcgaacc agcagcgtcg cgtcgggcca agcgaagcag acggcacggc atctctgtcg 2940

ctgcctctgg acccctctcg agagttccgc tccaccgttg gacttgctcc gctgtcggca 3000

tccagaaatt gcgtggcgga gcggcagacg tgagccggca cggcaggcgg cctcctcctc 3060

ctctcacggc accggcagct acgggggatt cctttcccac cgctccttcg ctttcccttc 3120

ctcgcccgcc gtaataaata gacaccccct ccacaccctc tttccccaac ctcgtgttgt 3180

tcggagcgca cacacacaca accagatctc ccccaaatcc acccgtcggc acctccgctt 3240

caaggtacgc cgctcgtcct cccccccccc ctctctacct tctctagatc ggcgttccgg 3300

tccatggtta gggcccggta gttctacttc tgttcatgtt tgtgttagat ccgtgtttgt 3360

gttagatccg tgctgctagc gttcgtacac ggatgcgacc tgtacgtcag acacgttctg 3420

attgctaact tgccagtgtt tctctttggg gaatcctggg atggctctag ccgttccgca 3480

gacgggatcg atttcatgat tttttttgtt tcgttgcata gggtttggtt tgcccttttc 3540

ctttatttca atatatgccg tgcacttgtt tgtcgggtca tcttttcatg cttttttttg 3600

tcttggttgt gatgatgtgg tctggttggg cggtcgttct agatcggagt agaattctgt 3660

ttcaaactac ctggtggatt tattaatttt ggatctgtat gtgtgtgcca tacatattca 3720

tagttacgaa ttgaagatga tggatggaaa tatcgatcta ggataggtat acatgttgat 3780

gcgggtttta ctgatgcata tacagagatg ctttttgttc gcttggttgt gatgatgtgg 3840

tgtggttggg cggtcgttca ttcgttctag atcggagtag aatactgttt caaactacct 3900

ggtgtattta ttaattttgg aactgtatgt gtgtgtcata catcttcata gttacgagtt 3960

taagatggat ggaaatatcg atctaggata ggtatacatg ttgatgtggg ttttactgat 4020

gcatatacat gatggcatat gcagcatcta ttcatatgct ctaaccttga gtacctatct 4080

attataataa acaagtatgt tttataatta ttttgatctt gatatacttg gatgatggca 4140

tatgcagcag ctatatgtgg atttttttag ccctgccttc atacgctatt tatttgcttg 4200

gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg ttacttctgc aggtcgactc 4260

tagaggatca aacattttta caacaattac caacaacaac aaacaacaaa caacattaca 4320

attacattta caattaccat ggcacagatt agaagcatgg cacagggcat tcaaacactt 4380

agcctcaata gcagcaacct ctccaagacg cagaagggtc cgctcgtgtc gaacagtttg 4440

ttctttggat ccaagaagct cacacagatc tcggcgaaga gtctgggggt gttcaagaag 4500

gacagcgtcc ttcgggtggt cagaaagtcc agctttcgga tttccgcaag cgtcgcaact 4560

gctgaggcac acggagcatc atctaggcct gcaaccgcac gcaagtcgag tggtctgtcg 4620

ggcacagttc ggatccccgg cgacaagtca atttctcata gatccttcat gtttggcgga 4680

cttgccagcg gcgagactag gatcacgggt ctcctggagg gcgaagatgt gattaacaca 4740

gggaaggcta tgcaagcaat gggagccagg atccgcaagg agggtgacac ttggatcatt 4800

gatggagtcg gtaacggagg tctgttggcg cctgaggctc ccctggactt cgggaatgcc 4860

gcgactggat gcaggttgac gatggggctc gtcggagttt acgacttcga ttcgactttt 4920

atcggcgatg cgagtctcac gaagcggcct atggggagag tgctgaatcc ccttcgggag 4980

atgggagtgc aggtcaagtc tgaagacggc gaccggctgc cggttaccct tagaggccca 5040

aagactccga cgccaatcac atatagagtg ccgatggctt cagcacaggt taagtctgcg 5100

gtgctcctgg ctggtctgaa cacaccgggc attaccacag tcatcgagcc aattatgact 5160

agggaccaca cggaaaagat gctgcagggc ttcggggcga atcttaccgt tgagacagac 5220

gctgatggcg tgaggactat cagactggaa ggaagaggca agctcacggg ccaagtgatt 5280

gacgtcccgg gggatccatc cagcactgcg tttcctctgg tggctgcact tttggtcccc 5340

ggctcagacg ttacgatctt gaacgtgctc atgaatccga ccaggacagg actcattctg 5400

acccttcagg agatgggtgc cgatatcgaa gtgattaacc caagactcgc gggcggggag 5460

gacgtcgctg atttgcgggt tagatcatct accctcaagg gagttacagt gcctgaggac 5520

agagcaccct ctatgatcga tgaataccct attctggctg tcgcagcagc tttcgcagag 5580

ggtgcaaccg tcatgaatgg cctggaggaa cttagggtta aggaatccga tagactcagc 5640

gcagtggcaa acgggttgaa gctcaacggc gtcgactgcg atgagggcga aacctcgttg 5700

gttgtgaggg gaagacctga cggcaaggga ctcggaaacg caagtggagc agcagtggca 5760

acgcacctcg atcataggat cgccatgtca ttcttggtca tgggcctcgt ttctgagaat 5820

ccggtcaccg ttgacgatgc aaccatgatt gccacatcct ttccagagtt tatggatttg 5880

atggcaggtc tgggggcgaa gattgaactg tcggatacca aggcggcgtg agtcgagcga 5940

atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 6000

gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 6060

tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 6120

tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 6180

tgtcatctat gttactagat cgggtttaaa ctatcagtgt ttgaaaaacg tacctattca 6240

ccccctctag gtgccctcaa tttataattt gacgataaat tgagcagata gatttattat 6300

ttttcgaaat taataatttt aaaaaacata taattagtat ttaaggtcaa aaaacactcc 6360

agagggtcca aaaattccaa gaaaatttat agagatattt tagatcatga gaaacttaaa 6420

taaagtattt agagttcatg aaaaatacat ttggaataat ctaaaactag atacgctcac 6480

ataagaatag ggaaaaaact ctagaataga tagaaaaatt cccaagaaga ttttcaagga 6540

taggttgata aacaatgaga acaaataata atttagaatg caagattttt ttgataagat 6600

tcaaataaaa gaattaaaaa tcaaggaatc tgatttgatt atctatacta tatttaaagc 6660

atcagtttca acggtcgtcg tgcgtcattt ttttacaaat aacccctcac agctatttta 6720

aattaatccg ttgcatatct ataaatgaca aacgacgccc aac 6763

<210> 2

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cagcacaggt taagtctg 18

<210> 3

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtctgtctca acggtaag 18

<210> 4

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cagttcccgt gcttgaag 18

<210> 5

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

caccatcgtc aaccactac 19

<210> 6

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcaatagcag caacctctc 19

<210> 7

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atagtcctca cgccatca 18

<210> 8

<211> 43

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctaatacgag tcactatagc gctcgagcgg ccgccgggga ggt 43

<210> 9

<211> 8

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acctcccc 8

<210> 10

<211> 27

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggatcctaat acgagtcact atagcgc 27

<210> 11

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctatagcgct cgagcggc 18

<210> 12

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggaactggca tgacgtgggt ttct 24

<210> 13

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cctgcccgtc accgagattt ga 22

<210> 14

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgtaaaacga cggccagt 18

<210> 15

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

caggaaacag ctatgacc 18

<210> 16

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tgaactgtcg gataccaagg cgg 23

<210> 17

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gttgggcgtc gtttgtcat 19

<210> 18

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tcctactact gtttaccatg cttg 24

<210> 19

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cctgcccgtc accgagattt ga 22

<210> 20

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tgaactgtcg gataccaagg cgg 23

<210> 21

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gttgggcgtc gtttgtcat 19

<210> 22

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tcctactact gtttaccatg cttg 24

<210> 23

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ggaactggca tgacgtgggt ttct 24

<210> 24

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

cagttcccgt gcttgaag 18

<210> 25

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ttccattgcc cagctatctg t 21

<210> 26

<211> 25

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

agagatagat ttgtagagag agact 25

<210> 27

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

cgacactctc gtctactcca a 21

<210> 28

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ctcaatagcc ctttggtctt 20

<210> 29

<211> 26

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

atactcaatt gtcctttaga ccatgt 26

<210> 30

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

agtgcagcgt gacccggtcg 20

<210> 31

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tcacaaccaa gcgaacaa 18

<210> 32

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

tttggtttgc ccttttcc 18

<210> 33

<211> 18

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

cttcgtgaga ctcgcatc 18

<210> 34

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ggcgaagatg tgattaacac 20

<210> 35

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gttgggcgtc gtttgtcat 19

Claims (11)

1. A nucleic acid molecule which is the sequence shown in SEQ ID NO.1 or a complementary sequence thereof.

2. The nucleic acid molecule of claim 1, comprising the following expression cassettes:

a first expression cassette for expressing an anti-glufosinate gene comprising the sequence shown as nucleotides 405-1945 of SEQ ID NO: 1; and

a second expression cassette for expressing a glyphosate-resistant gene, comprising the sequence shown by nucleotides 2266-6203 of SEQ ID NO. 1.

3. The nucleic acid molecule of claim 1 or 2, which is present in a maize plant, seed, plant cell, progeny plant or plant part.

4. A probe for detecting a maize transformant, which comprises a sequence represented by nucleotides 1 to 634 and/or nucleotides 5904-6763 of SEQ ID NO.1, or a complementary sequence thereof.

5. A primer pair for detecting a corn transformant, which can specifically amplify a sequence shown by nucleotides 1 to 634 or nucleotides 5904-6763 of SEQ ID NO.1 and a complementary sequence thereof.

6. The primer pair of claim 5, wherein the primer pair is:

i) a primer pair which specifically recognizes a sequence shown by nucleotides 1 to 634 of SEQ ID NO. 1;

ii) a primer pair which specifically recognizes the sequence shown by nucleotides 5904-6763 of SEQ ID NO. 1; or

) A primer pair which specifically recognizes the sequence represented by the nucleotides 1 to 634 of SEQ ID NO.1 and a primer pair which specifically recognizes the sequence represented by the nucleotides 5904-6763 of SEQ ID NO. 1.

7. The primer pair of claim 6, wherein the primer pair is a nucleotide sequence shown as SEQ ID NO. 18 and SEQ ID NO. 19; and/or the nucleotide sequences shown in SEQ ID NO:20 and SEQ ID NO: 21.

8. A kit or microarray for detecting maize transformants comprising the probe of claim 4 and/or the primer pair of any one of claims 5-7.

9. A method for detecting a maize transformant, comprising detecting the presence of the transformant in a test sample using:

-the probe of claim 4;

-a primer pair according to any one of claims 5 to 7;

-the probe of claim 4 and the primer pair of any one of claims 5-7; or alternatively

-a kit or microarray according to claim 8.

10. A method of breeding maize, the method comprising the steps of:

1) obtaining maize comprising the nucleic acid molecule of any one of claims 1 to 3;

2) subjecting the corn obtained in step 1) to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, selfing or crossing or a combination thereof to obtain seeds, plant cells, progeny plants or plant parts.

11. The method of claim 10, further comprising 3) identifying herbicide-resistant glufosinate ammonium and glyphosate in the progeny plant obtained in step 2), and detecting the presence or absence of the transformant therein using the method of claim 9.

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